A key aspect of cellular regulation that affords a number of potential opportunities for pharmacological intervention is the maintenance of protein homeostasis. Proteins are continuously being synthesized and degraded in cells, and their cellular levels must be strictly regulated. The ubiquitin-proteasome pathway is the major non-lysosomal proteolytic system in eucaryotic cells, consisting of a multi-enzyme cascade that modifies and degrades a diverse array of proteins, including many key regulatory molecules. The modification of cellular proteins by ubiquitin (Ub) and ubiquitin-like proteins (UBL) plays an essential role in a number of biological processes, including cell cycle control, transcriptional activation, signal transduction, antigen presentation, vesicular trafficking, and intracellular proteolysis. Ubiquitination is a dynamic reversible process, with a multitude of ubiquitin ligases (E3s) and deubiquitinases (DUBs) responsible for both the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes are responsible for maintaining adequate pools of free Ub within the cell and for regulating the ubiquitination status of cellular proteins. Defects in these enzymes appear to play a role in the pathogenesis of several diseases, including Parkinson's disease, Angelman's syndrome, cervical cancer, and von Hippel Lindau syndrome. This research proposal focuses on the DUBs and on the development of a novel high-throughput assay to identify agents that modulate the activity of these key regulatory enzymes. The assay being proposed is based on the observation that certain proteins require a defined and free amino-terminal residue in order to be functional. Fusions between Ub/UBL and these proteins will serve as substrates that undergo activation upon DUB cleavage. This reaction will be optimized and adapted to a high-throughput format for eventual use in screens for inhibitors/activators of clinically relevant DUBs.